Research Project: Reducing Impacts of Disease on Salmonid Aquaculture Production

Location: Office of The Director

2020 Annual Report

Objectives
Major impediments to production and profitability of U.S. aquaculture are the lack of genetically-defined species with traits for faster growth, greater feed efficiency/utilization, and improved disease resistance. Rainbow trout are important recreational and food fish species in the Great Lakes and it is thus important to improve productivity of this species in this region. Over these next 3 years we will focus on the following three Objectives and their supporting Sub-Objectives: Objective 1: Characterize mechanisms of innate immune response, and pathogen virulence, to control rhabdoviral diseases in salmonid aquaculture. • Sub-Objective 1.A.: Identify domains within viral proteins of infectious hematopoietic necrosis virus (IHNV) and viral hemorrhagic septicemia virus (VHSV) that interfere with the host virus recognition and response pathways in vitro. (Leaman, Stepien and Vakharia) • Sub-Objective 1.B.: Characterize the in vitro replication of recombinant IHNV and VHSV containing mutations designed to disrupt viral suppression of host recognition and response pathways. (Leaman and Vakharia) • Sub-Objective 1.C.: Assess the impact of IHNV and VHSV infection and subsequent innate immune suppression on the activation of dendritic cells (DCs). (Spear) • Sub-Objective 1.D.: Develop in vivo IHNV and VHSV challenge models in rainbow trout. (Spear and Shepherd) Objective 2: Use genetic techniques to characterize mechanisms of Flavobacterium virulence and identify potential strategies to control bacterial disease in salmonid aquaculture. • Sub-objective 2.A.: Develop genetic techniques for F. columnare strains that cause columnaris disease in rainbow trout. (McBride) • Sub-Objective 2.B.: Isolate and characterize F. columnare mutants and identify virulence factors associated with ability to cause disease in rainbow trout. (McBride) • Sub-Objective 2.C.: Develop improved genetic techniques for F. psychrophilum, the causative agent of bacterial coldwater disease. (McBride) Objective 3: Measure and modulate antimicrobial peptides (AMPs) as a means to control disease in salmonids. • Sub-Objective 3.A.: Characterize the environmental and endocrine contributions to regulation and expression of AMPs in rainbow trout. (Shepherd and Spear) • Sub-Objective 3.B.: Test the anti-viral and anti-bacterial activities of two synthetic trout AMPs in vitro. (Shepherd, Spear, Leaman and McBride)

Approach
For Objective 1: We will characterize the mechanisms of virulence for Viral Hemorrhagic Septicemia virus (VHSV) and Infectious Hematopoietic Necrosis virus (IHNV) in rainbow trout. Research will involve molecular analysis of viral diversity, mutational analysis of viral factors contributing to virulence in rainbow trout. These studies will utilize homologous in vitro systems (cell-lines and dendritic cells) to identify of host factors involved in recognition and response pathways to viral infection in rainbow trout. Lastly, disease challenge assays will be developed, and validated, to understand the virulence and the disease processes for IHNV and VHSV pathogens in rainbow trout. For Objective 2: This work will target mechanisms of pathogenesis of F. psychrophilum (causative agent in bacterial cold water disease) and F. columnare (causative agent in columnaris disease) in rainbow trout. To do this, we will use bacterial culture and genetic techniques to isolate mechanisms of pathogensis for Flavobacterium spp. in this species. Attenuated bacterial strains will be evaluated for pathogenesis using established disease challenge models, in this species. For Objective 3: We will characterize the physiological regulation of antimicrobial peptides (AMPs) and their actions in rainbow trout. To accomplish this, we will assess how environmental stressors, and hormones, affect expression (genes) and levels (proteins) of AMPs in this species. Additionally, we shall utilize in vitro techniques to evaluate biocidal actions of select AMPs against VHSV and IHNV and Flavobacterium spp.

Progress Report
This is a final report, which terminated December 2019. See the report for replacement project, 5090-31320-005-00D, “Reducing Impacts of Disease on Rainbow Trout Aquaculture Production”. The aim of Objective 1 is to characterize mechanisms of innate immune response, and pathogen virulence, to control rhabdoviral diseases in salmonid aquaculture. Infectious hematopoietic necrosis virus (IHNV) and viral hemorrhagic septicemia virus (VHSV) are highly contagious, pathogenic rhabdoviruses affecting fish in Europe and North America. Initial efforts of this objective focused on the expression of all IHNV gene products in cells to determine their impact on host cell gene expression using EPC (Epithelioma Papulosum Cyprini) cells and varying constitutively active promoter constructs. The IHNV M (matrix) protein was the most potent inhibitor of gene induction, mirroring what we have previously observed and reported for VHSV. The second component was to transition most of our in vitro work into using established Rainbow Trout (RT) cell-lines, and expand analyses of the matrix (M) and nucleoprotein (N) functions for INHV and VHSV. Culture and transfection procedures were optimized to enable use of RTG-2 (gonad) cells and RTgill-W1 cells. Findings suggest that the effects of M and the non-virion (NV) proteins of the INHV and VHSV pathogens remain the most robust modulators of host immune response across cell lines. Other viral genes studied, were the phosphoprotein (P) and glycoprotein (G), and results showed some more specific induction patterns that were linked to the cell type or dose of viral region used. The third component was to begin characterization of stress granules (SGs) in the host viral response pathway. Viral and bacterial infections stimulate host responses that are normally used to counter various forms of stress by inducing formation of SGs. Work in mammals shows that viral infection activates formation of SGs, but our understanding of these processes in fish is poor. In this last year, assays were developed to study the role of Stress Granules (SG), and stress-response signaling pathways, during infection in teleost cell-lines. Using the EPC fish cell-line (EPC) as a starting point, we observed that the native Great Lakes type of the vial hemorrhagic septicemia virus VHS suppressed the host antiviral response by inhibiting production of most new proteins within the cells, except for production of viral proteins. Infection of EPC fish cells with a Great Lakes VHS virus, not containing the NV region, exhibited lower levels of viral protein synthesis despite increased levels of viral messenger ribonucleic acid (increased viral gene abundance). These findings show a subtle role for the NV region in Great Lakes type of VHS virus mediated pathogenesis via alteration of host protein shutoff. These comparative approaches, using various cell-lines, have allowed a glimpse into the complex nature of novirhabdoviral pathogenetic strategy across a variety of cells and teleost hosts. Information from this work may enable identification of new viral targets that modulate the host-pathogen interaction, and immunogenicity, which could be adopted to design more efficient vaccination strategies. The aim of Objective 2 is to use genetic techniques to characterize mechanisms of Flavobacterium virulence and identify potential strategies to control bacterial disease in salmonid aquaculture. Bacterial cold-water disease (caused by Flavobacterium psychrophilum: Fp ), and columnaris disease (caused by Flavobacterium columnare: Fc) are major problems for salmonid aquaculture. Little is known regarding the virulence factors involved in these diseases, and control measures are inadequate. To address this problem, techniques have been developed to enable deletion of various genes to identify genetic controls that contribute to the virulence of these pathogens. Proteins secreted by a number of genetic systems are likely virulence factors, and targets for the development of control. We have tested 43 Fc strains and identified 11 that could be genetically manipulated, and two Fc strains were selected for genetic studies. We constructed plasmids to delete the gldN gene (gliding motility protein) from two Fc strains. gldN is required for secretion of protein toxins and virulence in Flavobacterium spp. The deletion plasmid was introduced into both model Fc strains and gldN deletion mutants were obtained, and were deficient in protein secretion and in gliding motility. In vivo testing showed that virulence was reduced in the gldN mutants compared with wild-type or re-complimented Fc strains. As expected, deletion of gldN, which encodes a component of the type 9 (IX) secretion system (T9SS), resulted in loss of virulence. Pursuing this idea further, Liquid Chromatography-Mass Spectroscopy (LCMS) was used to identify possible toxins that contribute to virulence. As for secreted non-toxic proteins thought to be involved with virulence, genes encoding three secreted proteases and two secreted chondroitinases were identified and deleted. Single gene deletions had little effect on virulence against zebrafish, but a strain lacking all five of these genes exhibited greatly reduced virulence in zebrafish. In this vein, another deletion mutant for the cell surface adhesin (sprF) protein (needed for secretion of the motility adhesin SprB to the cell surface) was constructed to test in rainbow trout to see if they are deficient for the enzymes listed above and determine if these strains can function as protective vaccines. We also conducted research on a Fc protease secretion (porV) deletion mutants (defective for secretion but not motility) and complemented strain to separate motility defects and secretion defects as causes for decreased virulence of Fc mutants. We also began our proposed genetic manipulation of Fp, and transferred plasmid into two Fp strains, for comparable pathogenicity research. Compared to wild-type and complemented strains, the gldN mutant was deficient in adhesion, gliding motility, and in extracellular proteolytic and hemolytic activities. The gldN mutant exhibited reduced virulence in rainbow trout and complementation restored virulence, suggesting that the T9SS plays an important role in the disease. Given strong international interest in combatting Fp pathogenesis, these strains are being analyzed by international collaborators in France to examine the virulence of Fp wild type and mutant cells in rainbow trout using an immersion challenge. The aim of Objective 3 is to measure and modulate antimicrobial peptides (AMPs) as a means to control disease in salmonids. AMPs are an ancient part of the innate immune system, which is a first line of defense against pathogens. We have identified 6 AMP genes in and mapped their locations to specific chromosomes in the rainbow genome. Using RNA sequencing, we showed that levels of these AMPs were altered by aquaculture stressors (handling stress, salinity, temperature and re-use water) and Fp bacterial challenge in rainbow trout. To further this work, synthetic peptide cores were designed and synthesized for use in studies to assess their biocidal actions against flavobacterial pathogens and their biofilms, in vitro. Initial work in Fc has not been entirely successful at this point as assays, and proteins, need further optimization. We have also evaluated how the stress steroid, cortisol, affects the liver immune response in rainbow trout as the immune response of the liver in finfish is poorly understood. To address this, we evaluated the short-term effects of lipopolysaccharide (LPS: a pathogen mimetic) and cortisol on the liver immune response in rainbow trout. Abundance of two AMP mRNAs were elevated with LPS and suppressed by cortisol. We believe this is among the first reports showing a direct effect of cortisol on the liver immune response in a finfish and understanding how it affects host immunity will inform improved husbandry practices that alter AMP levels (reduced acute stress) to improve animal health. As part of a legacy project (5090-31320-003-00D, Accession # 0429302), we have sequenced the yellow perch (Perca flavescens) genome. The yellow perch genome is estimated to be 0.9-1.1 Gb in size and is comprised of approximately 31,500 genes. The quality of the genome assembly, and annotation, assessed by running the Benchmarking Universal Single-Copy Orthologs (BUSCO) program, indicate a high-quality genomic assembly for yellow perch. Using this resource, we are annotating a novel RNA-sequencing dataset to study the effects of gender on the immune response in this fish species. With regard to this, recent work is showing gender-specific differences in gene splicing and abundances of various genes in yellow perch treated with a pathogen mimetic called lipopolysaccharide (LPS). We have preliminary data that suggests that mRNA abundances of the AMP hepcidin is upregulated in both male and female yellow perch, but in females hepcidin mRNA levels return to base-line faster than males, which underscores sex-specific differences in immune response in this species.

Accomplishments
1. New genomic resources developed for yellow perch, Perca flavescens. Yellow perch is a freshwater fish in high demand for human consumption and, consequently, numbers in the wild have been substantially reduced due to overfishing and other factors. Modern genomic resources are needed for conservation genetics and for genetic improvement programs for the aquaculture industry. To address this, ARS researchers in Milwaukee, Wisconsin, and researchers from The Ohio State University (Piketon, Ohio) sampled yellow perch from eight populations to develop libraries consisting of two types of genomic markers called simple sequence repeats and single nucleotide polymorphisms. This research resulted in the identification of thousands of new genetic markers distributed throughout the yellow perch genome. These genetic markers are important genomic resources that will enable scientists to increase the rate of genetic gain for traits of interest in yellow perch aquaculture, and will enhance efforts aimed at conserving genetic diversity within and among wild yellow perch populations.

2. The nonvirion region, is part of the viral hemorrhagic septicemia virus (VHSv) gene, but encodes for this non-structural protein. (VHSv) has been found to affect the host antiviral response in unexpected ways. VHSv is one of the most deadly infectious fish pathogens posing a serious threat to the aquaculture industry. As with related viruses, the Great Lakes VHSv contains a unique and highly variable nonvirion gene, which is thought to enable infection of host cells and its ability to evade the host immune response. To elucidate function of the nonvirion gene, ARS researchers in Milwaukee, Wisconsin, and researchers from the University of Toledo and Wright State University (Ohio) used a fish cell-line and observed that the native Great Lakes type of VHS virus suppressed the host antiviral response by inhibiting production of most new proteins within the cells, except for production of viral proteins. Infection of the fish cell line with a Great Lakes VHS virus, not containing the nonvirion region, exhibited lower levels of viral protein synthesis despite increased levels of viral expression. These findings show a subtle role for the nonvirion region in Great Lakes type of VHS virus mediated pathogenesis via alteration of host protein shutoff. Information from this work may enable identification of new viral targets that modulate the host-pathogen interaction, and immunogenicity, which could be adopted to design more efficient vaccination strategies.

Review Publications
Guo, L., Yao, H., Shepherd, B.S., Sepulveda-Villet, O.J., Zhang, D., Wang, H. 2019. Development of a genomic resource and identification of nucleotide diversity of yellow perch by RAD sequencing. Frontiers in Genetics. https://doi.org/10.3389/fgene.2019.00992.
Kesterson, S.P., Ringiesn, J., Vaharia, V.N., Shepherd, B.S., Leaman, D.W., Krishnamurthy, M. 2020. Effect of the viral hemorrhagic septicemia virus nonvirion protein on translation via the PERK – eIF2alpha pathway. Viruses. 12(5), 499. https://doi.org/10.3390/v12050499.